The traditional view that the adult heart has no capacity for regeneration or cardiomyogenesis has been called into question, and multiple lines of evidence now support some albeit limited potential for cardiac regeneration. However, there is a fundamental gap in our understanding of the physiological processes and pathways that regulate this regenerative capacity. This gap represents an important problem because it limits our ability to exploit the heart's regenerative capacity to restore cardiac function in disease. Th long-term goal is to understand the mechanisms regulating cardiac regeneration and to learn how to control it. The objective of the current application is to understand the mechanisms and contribution of exercise-induced cardiomyogenesis. Preliminary data produced in the applicants' laboratories suggest that exercise induces a potentially regenerative response in the heart. These studies also identify a new transcriptional pathway, including the co-activator, CITED4, as a key mediator of the cardiac exercise response. This proposal is based on three central hypotheses: 1) that exercise enhances the endogenous regenerative capacity of the heart; 2) that CITED4 is necessary for exercise-induced cardiomyogenesis; and 3) that CITED4-dependent cardiomyogenesis contributes to the benefits of exercise at baseline and in disease. The rationale for the proposal is that understanding the physiological mechanisms regulating the heart's endogenous regenerative capacity will advance efforts to therapeutically harness this capacity for clinically important conditions associated with loss of cardiomyocytes. The central hypotheses will be tested in three integrated Specific Aims. In Aim 1, a recently developed technology that combines genetic fate mapping with mass spectrometry-based imaging (Multi-isotope Imaging Mass Spectrometry or MIMS) will be used to unambiguously identify the number, source, and fate of new cardiomyocytes formed in two models of exercise. In Aim 2, the consequences of lineage-specific CITED4 inactivation in cardiomyocytes and c-kit-expressing cardiac progenitor cells will be determined at baseline and in exercise. In Aim 3, the ability of exercise-induced cardiomyogenesis to mitigate remodeling after myocardial infarction and whether CITED4 is required will be examined. In vivo studies will be supported by in vitro investigation of isolated cell populations including adult cardiomyocytes and c-kit-expressing cardiac progenitor cells to elucidate the underlying mechanisms. The approach combines innovative hypotheses, technologies, and unique animal models with the complementary expertise of an outstanding team of collaborating investigators. The proposed research is significant, because it is expected to advance our understanding of cardiac regeneration and the benefits of exercise as well as their potential to mitigate disease. These studies will provide new insights into the physiological mechanisms controlling cardiac growth and regeneration as well as informing therapeutic approaches to diseases associated with cardiomyocyte loss such as heart failure and ischemic heart disease.